The long-term goal of this R01 application is to elucidate the role(s) of secreted phospholipase A2 (PLA2) in Rickettsia prowazekii pathobiology. R. prowazekii is an obligate intracytoplasmic parasite and the etiological agent of louse-vectored epidemic typhus in humans. Rickettsial derived PLA2 is regarded as essential to pathogenesis and is thought to mediate entry into the host cell, escape from the phagosome, and lysis of the host cell - yet its identity remains unknown. The R. prowazekii genome encodes two putative members of the patatin superfamily of phospholipase/acyl hydrolase enzymes, RP534 and RP602 that are the focus of this application. In Aim I we will test the hypothesis that R. prowazekii secretes RP534 and RP602 during infection of eukaryotic host cells. We provide preliminary evidence showing that both RP534 and RP602 are activated in a secretion-dependent fashion. We will determine if rickettsiae secrete these proteins to the culture medium;into host cell cytosol;to the rickettsial cell surface where they remain tethered;or any combination thereof. In Aim II we will test the hypothesis that (A) RP602 plays a role in host cell entry and (B) RP534 plays a specialized role in R. prowazekii pathogenesis. RP602 is the only known PLA2 that is common to all sequenced rickettsial genomes whereas RP534 is not. In part (A), we will determine if RP602 is the long-sought after rickettsial PLA2 that has been implicated in mediating entry into eukaryotic host cells. We will use antibody and inhibitor studies to examine its role in host cell entry as a potential target for therapeutic intervention. We will also elucidate the mechanism of RP602 secretion-dependent activation. In part (B) we will determine the role of RP534 in rickettsial pathogenesis. We present preliminary data showing that RP534 works to dampen activation of caspase-1-mediated inflammatory responses using macrophage-like cells in culture. We will inhibit RP534 activity during rickettsial infection of primary macrophages to examine its role in modulating caspase-1 activation. We will also inhibit RP534 activity during infection of primary endothelial cells to uncover other possible roles of RP534 in host cell infection. Defining the role of PLA2 enzymes in rickettsial pathogen-host interactions could unveil novel targets for the development of new anti- rickettsial therapeutics to combat this Select Agent. Anticipated future studies will involve uncovering a role for rickettsial PLA2 in modulation of host cell signal transduction pathways. PUBLIC HEALTH RELEVANCE: Rickettsia prowazekii is the louse-vectored agent of epidemic typhus in humans and is designated as a re- emerging pathogen and category B Select Agent. The focus of this application is to characterize the role of secreted rickettsial phospholipase A proteins in rickettsial entry into a eukaryotic host cell. In addition, screening strategies will be employed to identify other secreted rickettsial proteins that could play important roles in defining host-pathogen interactions and could represent novel targets for the development of novel anti-rickettsial therapeutics.